In this revised PPG, we propose to analyze the function of a new family of genes that we have recently discovered called TIM (T cell Immunoglobulin and Mucin domain containing molecules), which was initially shown to be expressed in T cells and play a crucial role in regulating immunity and tolerance. However, recent data suggests that TIM molecules are not only expressed on T cells but also expressed on antigen presenting cells (APC) in the peripheral immune compartment and in target tissues. The role of TIM molecules in regulating the function of T cells vs. APCs is not well understood. Preliminary data suggest that the TIM family of molecules can positively or negatively costimulate T cell activation and also promote APC activation. Based on these observations, we hypothesize that this family of molecules may have a very important role in regulating both innate and adaptive immune systems and could be exploited in a number of diseases and inflammatory conditions, including autoimmunity, asthma, transplant rejection and tumor immunity. This PPG brings together 3 research projects and 3 cores, involving 11 independent investigators, to address the role of the TIM family of molecules in autoimmunity and transplant rejection. The proposed studies will utilize and examine animal models and human disease conditions. The three projects are highly interdependent and address a common theme: What is the role of the TIM family of molecules in regulating immunity and tolerance? The main themes of the three projects are: 1. The roles of the TIM family of genes in regulating autoimmune T cell responses in mouse models (Project I: Vijay Kuchroo and Joan Goverman, PIs);2. TIM- 3 regulation of immune responses in the CNS in humans (Project II: David Hafler, David Anderson and Larry Kane PIs);3. TIM proteins in regulation of transplantation tolerance (Project III: Terry Strom, Wenda Gao and Larry Kane PI). To understand the mechanism by which TIM protein mediate their function in adaptive and innate immune system, in the revised grant we have included a new co-PI (Larry Kane) on projects 2 and 3 to analyze the signaling pathways induced by TIM molecules. The three cores (Administrative, Antibody/lg- fusion protein, Transgenic/Knock-out core) will support these three projects and provide tools that will be shared by all the projects, thus accelerating discovery in an efficient and cost effective manner. Lay Summary: By sharing a set of common set of standardized reagents and information, and conducting complementary experiments in animal models and humans and among different types of inflammatory diseases, the PPG will build a comprehensive understanding of the role of the TIM family of genes in regulation of immunity and tolerance with the potential of exploiting this pathway for therapeutic purposes in humans. PROJECT 1: Role of TIM of Genes in Regulating Autopathogenic T Cell Responses PROJECT 1 DESCRIPTION (provided by applicant): The recently discovered Tim (T cell Immunoglobulin and Mucin domain) gene family has emerged as an important player in immune regulation. We identified Tim-3 as a molecule expressed specifically on CD4+ Th1 but not Th2 cells. The interaction of Tim-3 with its ligand, galectin-9, triggers cell death in Th1 cells, thereby dampening Th1 immunity. Tim-3 is also expressed on CD8+ Tc1 but not Tc2 cells. However, whether Tim-3 similarly regulates Tc1 effector cells has not been examined. Interestingly, we have now found that Tim-3 is expressed on all dendritic cells (DCs), the major antigen presenting cells (APCs) of the immune system and on central nervous system (CNS) microglia, the local APCs in the CNS. These observations raise the possibility that Tim-3 may regulate the adaptive immune response via its expression on APCs. Indeed, we have found that Tim-3, contrary to its role in dampening Th1 immunity via its expression on Th1 cells, may promote Th1 immunity via its expression on APCs. This suggests that Tim-3 may serve opposing roles in the innate and adaptive immune systems. Tim-1 is expressed on all activated CD4+ T cells with Th2 cells expressing slightly higher levels relative to Th1 cells. Tim-1 can function as a costimulatory molecule and appears to be important in the induction of T cell tolerance. These observations support an important role for Tim-1-mediated regulation of CD4+ T cell responses. However, the effects of Tim-1 in Th1 cells, and the newly identified subset of Th-17 cells, have not been examined. In addition, we have found that Tim-1 is expressed on all CD8+ T cells directly ex vivo, raising the possibility that Tim-1 may regulate CD8+ T cell responses. We have developed several new antibodies, fusion proteins and transgenic and knock-out mice that will allow for the first time a detailed investigation of the role of Tim-3 in DCs, CNS microglia and CD8+ Tc1 cells and Tim-1 in Th1, Th-17 and CD8+ T cells. Using these tools, we propose to 1) Determine the function of Tim-3 in the innate immune system specifically on DCs and CNS derived microglial cells, 2) Define the role of Tim-3 in the generation and regulation of self-reactive CD8 T cells, 3) Determine the role of Tim-1 signaling in the generation and effector function of encephalitogenic T cells including Th1, Th-17 and CD8+ T cells. The studies proposed here will analyze the role of Tim-3 and Tim-1 in the regulation of autoimmune responses by affecting the function of DCs, CNS microglia, Th-1, Th-17, and CD8+ T cells, the subsets of immune cells in which the functional role of Tim-1 and Tim-3 is not known.